MOLECULAR BASIS OF HUMAN CYTOCHROME P450 3A FUNCTION

描述(逐字摘自申请者摘要)：长期目标 这项建议中所描述的研究的目的是阐明结构基础 针对人细胞色素P450 3A的底物特异性和协同性。 这些酶是用途非常广泛的催化剂，在 多种药理和毒理化合物的代谢 利息。CYP3A4是大多数人类肝脏中表达最高的P450， 对临床上使用的药物的新陈代谢负责比其他任何药物 P450，是许多严重的药物相互作用的中心。CYP3A5是 在大约四分之一的人的肝脏中表达。3A4和3A5 显示84%的氨基酸序列相同，并代谢许多相同的氨基酸 底物。然而，每种酶都会产生一种不同的代谢产物模式 某些药物，如环孢菌素A和咪达唑仑。一个有趣的问题是 这些酶是如何接受如此多结构不同的底物的 对单一化合物表现出显著的区域和立体选择性。 CYP3A4和3A5与某些底物也表现出正的协同作用， 其表现为自激活(同质协作性)或 被第二种化合物激活，如α-萘黄酮(异构性 协作性)。在其他情况下，CYP3A4可以容纳两种底物 对彼此的新陈代谢没有明显影响。在以下期间生成的结果 目前的获奖期使我们能够识别出许多氨基酸 决定CYP3A4底物专一性和协同性的残基。这个 拟议研究的中心假设是非典型相互作用 (激活、部分抑制、无抑制)两种细胞色素P3A4底物 反映同时占用两个或更多首选位置 单人大捆绑口袋。这将通过以下组合进行测试 不同底物的定点诱变功能分析 效应器、核磁共振波谱和3-D分子 模特儿。具体目标是：1)确定 CYP3A4的同向性和异构性协同性；2)决定了 人细胞色素P3A氧化典型药物底物的结构基础 3)用核磁共振法确定酶活性部位的底物取向； 4)确定所选化合物抑制细胞色素P3A的结构基础。 了解人类P450 3A功能的分子基础应该能够 这些酶的底物、激活剂和抑制物的预测，使 有可能将药物之间的相互作用和个体间差异降至最低 在药物代谢方面。

DESCRIPTION (Verbatim from the Applicant's Abstract): The long-term objective of the research described in this proposal is to elucidate the structural basis for the substrate specificity and cooperativity of human cytochromes P450 3A. These enzymes are very versatile catalysts and play a crucial role in the metabolism of a wide variety of compounds of pharmacological and toxicological interest. CYP3A4 is the most highly expressed P450 in the liver of most humans, is responsible for the metabolism of more clinically used drugs than any other P450, and is the locus of numerous serious drug-drug interactions. CYP3A5 is expressed in the liver of approximately one in four individuals. 3A4 and 3A5 exhibit 84 percent amino acid sequence identity and metabolize many of the same substrates. However, each enzyme produces a distinct pattern of metabolites of certain drugs such as cyclosporin A and midazolam. An intriguing question is how these enzymes can accept so many structurally diverse substrates yet exhibit remarkable regio- and stereoselectivity towards a single compound. CYP3A4 and 3A5 also exhibit positive cooperativity with certain substrates, which manifests itself as autoactivation (homotropic cooperativity) or activation by a second compound, such as alpha-naphthoflavone (heterotropic cooperativity). In other cases, two substrates can be accommodated by CYP3A4 with no apparent effect on each others' metabolism. Results generated during the current award period have allowed us to identify many of the amino acid residues responsible for substrate specificity and cooperativity of CYP3A4. The central hypothesis of the proposed studies is that atypical interactions (activation, partial inhibition, no inhibition) between two CYP3A4 substrates reflect simultaneous occupancy of two or more preferred locations within a single large binding pocket. This will be tested by a combination of site-directed mutagenesis functional analysis with a variety of substrates and effectors, nuclear magnetic resonance (NMR) spectroscopy, and 3-D molecular modeling. The Specific Aims are to: 1) determine the structural basis for homotropic and heterotropic cooperativity of CYP3A4; 2) determine the structural basis for oxidation of prototypical drug substrates by human CYP3A enzymes; 3) determine substrate orientation in the CYP3A4 active site by NMR; 4) determine the structural basis for CYP3A inhibition by selected compounds. Knowledge of the molecular basis of human P450 3A function should allow the prediction of substrates, activators, and inhibitors of these enzymes, making it possible to minimize drug-drug interactions and interindividual differences in drug metabolism.